Thermo-sensitive chitosan colloid, preparation method and application of thermo-sensitive chitosan colloid in regulation and control of water-in-water emulsion type

Abstract

The invention provides a thermo-sensitive chitosan colloid, a preparation method and application of the thermo-sensitive chitosan colloid in regulation and control of water-in-water emulsion types, N-isopropylacrylamide (NIPAM) is grafted to a chitosan side chain to obtain a polymer with thermo-sensitivity, and then the polymer is induced to form thermo-sensitive responsive chitosan polymer colloid particles (CS-g-PNIPAM CPs) through an external environment. Compared with the prior art, the method has the advantages that the molecular structure is not changed, the hydrophilicity and hydrophobicity of the colloid are changed through external environment stimulation, and the emulsion type is regulated and controlled.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of high polymer materials and the field of emulsion, and particularly relates to a temperature-sensitive chitosan colloid, a preparation method thereof and application of the temperature-sensitive chitosan colloid in regulating and controlling a water-in-water emulsion type. BACKGROUND

[0002] An emulsion is a thermodynamically unstable dispersion formed by one liquid in the form of microdroplets dispersed in another liquid under the action of mechanical force or other external force. In a traditional emulsion, the two immiscible phases are oil phase and water phase, and according to the dispersed phase, the emulsion can be divided into water-in-oil (W / O), oil-in-water (O / W), water-in-oil-in-water (W / O / W), oil-in-water-in-oil (O / W / O) and various types of emulsions.

[0003] In addition to the traditional oil-water emulsion, two immiscible aqueous solutions can also produce an emulsion, namely, a full aqueous phase emulsion, also known as a water-in-water emulsion or a double aqueous phase emulsion, which is a water-water dispersion system formed by using the spontaneous discrete phase separation behavior of polymers and polymer / inorganic salt in an aqueous solution. Since the full aqueous phase emulsion does not contain an oil phase, it has the characteristics of non-toxicity, green environmental protection and better biocompatibility, and avoids more toxic ingredients in advanced applications in cosmetics, food, biotechnology and drug delivery.

[0004] Most of the current researches focus on the stabilizers of the double aqueous phase emulsion and the application of the emulsion, and there are few studies on the stability mechanism and type regulation of the double aqueous phase emulsion. The stability factors of the double aqueous phase emulsion generally include the molecular weight and concentration of the polymer, the type, shape and size of the colloidal particles, the pH value, temperature and ionic strength of the dispersed liquid; for the double aqueous phase emulsion, the current research believes that the double aqueous phase emulsion constructed when the colloidal particles have stronger affinity with the continuous phase will have better stability, and the type of the emulsion is determined by the volume ratio of the two phases, the phase with larger volume is the external phase, and the phase with smaller volume is the internal phase. Responsive emulsions are widely used in various pharmaceutical applications, such as nano-carriers and thermal gel drug delivery, and their applications are not limited to drug delivery, but also applied to biomedical applications, such as chromatography systems and cell culture applications. Generally, the substance delivery is wrapped in the internal phase, so the type of the emulsion can be regulated to realize the large volume in the internal phase to increase the delivery amount. In 2018, the Binks research group tried to regulate the type of the emulsion by changing the hydrophilicity and hydrophobicity without changing the volume ratio, but it was not realized. SUMMARY

[0005] The present application aims to provide a temperature-sensitive chitosan colloid and a preparation method thereof, in which N-isopropyl acrylamide (NIPAM) is grafted to the side chain of chitosan to obtain a temperature-sensitive chitosan polymer (CS-g -PNIPAM), by the regulation of the environment, inducing the formation of temperature-sensitive responsive chitosan polymer colloidal particles (CS-g - PNIPAM CPs), the preparation method is simple and efficient.

[0006] The present application also aims to provide a temperature-sensitive chitosan colloidal application in regulating water-in-water emulsion type, which changes its hydrophilic and hydrophobic properties by external environmental stimulus (temperature), as an emulsifier to regulate the emulsion type, and also has a stabilizing effect on the whole water phase emulsion. The present application realizes the regulation of the emulsion type by only external environmental stimulus without changing its molecular structure.

[0007] The specific technical scheme of the present application is as follows:

[0008] A preparation method of a temperature-sensitive chitosan colloidal, comprising the following steps:

[0009] 1) Mix N-isopropyl acrylamide, azobisisobutyronitrile and isopropyl alcohol, add chain transfer agent and stir under the atmosphere of nitrogen, heat and react, after the reaction is completed, dialysis, freeze-drying, to obtain carboxyl-terminated poly-N-isopropyl acrylamide PNIPAM-COOH;

[0010] 2) Dissolve chitosan in acetic acid solution to obtain a chitosan solution, then add PNIPAM-COOH and condensing agent, stir and react at room temperature, after the reaction is completed, dialysis, freeze-drying, to obtain temperature-sensitive chitosan CS-g - PNIPAM;

[0011] 3) Dissolve CS-g - PNIPAM in acetic acid solution, stir and adjust the pH to appear Tyndall phenomenon, to obtain temperature-sensitive chitosan colloidal particles CS-g - PNIPAM CPs.

[0012] The N-isopropyl acrylamide in step 1) is refined N-isopropyl acrylamide NIPAM, the specific method is: dissolved in n-hexane at 60-65℃, hot-filtration, and placed in a fume hood until the n-hexane is completely volatilized;

[0013] The azobisisobutyronitrile AIBN is dissolved in anhydrous ethanol at 40-50℃ for recrystallization before use, and placed in a fume hood for volatilization;

[0014] In step 1), the mass ratio of the N-isopropyl acrylamide and azobisisobutyronitrile is 100:1-20:1; the amount ratio of azobisisobutyronitrile and isopropyl alcohol is 1:800-1:300 g / mL;

[0015] The ratio of azobisisobutyronitrile to chain transfer agent added in step 1) is 3:1-1:3 mg / μL; the chain transfer agent is mercaptopropionic acid;

[0016] The heating reaction in step 1) is at 50-80℃ for 8-15h;

[0017] The dialysis in step 1) is performed in ultrapure water, and dialysis is performed for 3-7 days using a dialysis bag (MD: 3400) to remove organic solvents.

[0018] In step 2), the chitosan is dissolved in an acetic acid solution with a volume fraction of 0.5-3% to obtain a chitosan solution with a concentration of 0.5-5 mg / mL;

[0019] In step 2), the mass ratio of chitosan to PNIPAM-COOH is 1:4-4:1;

[0020] In step 2), the mass ratio of chitosan to condensing agent is 1:4-4:1; the chitosan has a low viscosity, <200 mPa·s;

[0021] In step 2), the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC / NHS), and the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:3-3:1;

[0022] In step 2), the stirring reaction at room temperature is performed for 24h;

[0023] In step 2), the dialysis is performed in ultrapure water, and dialysis is performed for 3-7 days using a dialysis bag (MD: 3400) to remove organic solvents.

[0024] The dialysis in the present application is performed for 3-7 days, and the purpose of dialysis is to remove organic solvents (isopropyl alcohol or acetic acid) and retain the synthesized product. Organic solvents are toxic, but the stabilizer of the full aqueous emulsion prepared in the present application is non-toxic and environmentally friendly, and the organic solvents need to be removed to ensure the advantages of the preparation and application of the aqueous emulsion.

[0025] In step 3), the organic solvent is an acetic acid solution with a volume fraction of 0.5-3%;

[0026] In step 3), the CS-g - The PNIPAM is dissolved in an organic solvent to a concentration of 0.5-5 mg·mL -1 .

[0027] In step 3), the pH of the solution is adjusted to 6.0 using NaOH solution under stirring conditions to change the weak intermolecular interaction forces (hydrogen bonds, van der Waals forces, hydrophilic and hydrophobic interactions, etc.) to induce aggregation and self-assembly to form a colloid, thus obtaining CS-g-PNIPAM CPs.

[0028] The present invention provides a temperature-sensitive chitosan colloid, which is prepared by the above method, and the obtained colloid particle size is 100nm-500nm.

[0029] The method for preparing a thermosensitive chitosan colloidal stabilizer according to this invention is as follows: First, N-isopropylacrylamide is modified to obtain carboxyl-terminated poly-N-isopropylacrylamide. Chitosan is dissolved in acetic acid solution to obtain a chitosan solution. Then, the carboxyl-terminated poly-N-isopropylacrylamide is added to the chitosan solution to react, introducing thermosensitive groups onto the chitosan, thus obtaining thermosensitive chitosan. The synthesized thermosensitive chitosan is dissolved in acetic acid solution. After complete dissolution, the pH is adjusted to 6.0 using NaOH solution under magnetic stirring, thereby changing the intermolecular interaction forces and causing self-assembly and aggregation to obtain CS-g. - PNIPAM CPs. Using the prepared CS-g - PNIPAM CPs stabilizers are reassembled at the water-water interface under homogenization. By changing the temperature, a stable all-aqueous emulsion is formed, thus controlling the emulsion type.

[0030] This invention provides an application of thermosensitive chitosan colloid in regulating the type of water-in-water emulsion, wherein the thermosensitive chitosan colloid is used as an emulsifier to regulate the type of emulsion.

[0031] The specific application method is as follows:

[0032] Polyethylene glycol is dissolved in a thermosensitive chitosan colloidal solution and heated to the corresponding temperature to obtain a polyethylene glycol solution; dextran is dissolved in ultrapure water, its pH value is adjusted, and it is heated to the same temperature as the polyethylene glycol solution to obtain a dextran solution; the polyethylene glycol solution and the dextran solution at the same temperature are mixed and allowed to stand until the mixture forms two macroscopic phases that separate into layers; under homogenization, it is made into an all-aqueous emulsion (or a water-in-water emulsion).

[0033] The molecular weight of the polyethylene glycol (PEG) is 20,000;

[0034] The molecular weight of the dextran Dex is 200,000;

[0035] The polyethylene glycol concentration in the polyethylene glycol solution is 10-30 wt%.

[0036] The concentration of dextran in the dextran solution is 10-30 wt%.

[0037] The volume ratio of the mixed polyethylene glycol solution and dextran solution is 1:3-3:1.

[0038] The temperature-sensitive chitosan colloidal solution is obtained by dissolving temperature-sensitive chitosan colloids in an acetic acid solution with a volume fraction of 0.5-3%, and the concentration of the temperature-sensitive chitosan colloids is 0.5-5.0 mg·mL -1 .

[0039] The dextran solution is obtained by adjusting the pH value and heating to the same temperature as the polyethylene glycol solution, and the temperature is 25-55 DEG C, preferably, the controlled temperature is 25 DEG C, 37 DEG C, 45 DEG C and 55 DEG C respectively, and the pH value is adjusted to 6.0 by using NaOH solution.

[0040] The homogenization is specifically performed at a homogenization speed of 6000-10000 r·min -1 , and a homogenization time of 1-5 min.

[0041] The temperature-sensitive chitosan colloids provided by the application are prepared by grafting temperature-sensitive groups to the side chains of chitosan to prepare temperature-sensitive chitosan (CS-g - PNIPAM), which can be dissolved in an organic solvent, the interaction force of the molecules is changed by changing the solution temperature, and self-assembly is induced by hydrophobic interaction to form responsive temperature-sensitive chitosan colloids (CS-g - PNIPAM CPs), which can be used as emulsifiers to stabilize all-water-phase emulsions. The performance of the formed CS-g - PNIPAM CPs is highly controllable, and the type of emulsion can also be controlled by changing the temperature. The multifunctional all-water-phase emulsion has potential application prospects in the fields of food, cosmetics, biomedicine and the like.

[0042] The existing technology is to control the type of emulsion by controlling the volume ratio of the emulsion, compared with the prior art, the application does not change the molecular structure, but changes the hydrophilicity and hydrophobicity of the colloids by external environment stimulation to control the type of emulsion. The application is based on an all-water-phase system, and the all-water-phase emulsion can be used for the research and development of low-calorie functional food, micro-chemical reactors in food and biological processing technology, and can be used as active carriers in biological drugs and oil-free cosmetics; and can be used in the functional application research in the fields of artificial cell interface assisted design, simulation cell material, cell and biocompatible particle preparation and 3D biological printing new material research and development. The application can also realize the control of the type of emulsion by changing the external environment, can realize the high loading capacity of the all-water-phase emulsion for active substances, and expands the application field of the all-water-phase emulsion. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The CS-g- Transmission electron microscope image of PNIPAAM CPs;

[0044] Figure 2 Fluorescence microscope image of stable whole aqueous phase emulsion prepared by temperature-sensitive chitosan colloids at pH 6.0, 45℃, after 4h standing; the volume ratio of two phases V - Contact angle picture of PNIPAAM with PEG water phase and Dex water phase at pH 6.0;

[0045] Figure 3 Fluorescence microscope image of stable whole aqueous phase emulsion prepared by temperature-sensitive chitosan colloids at pH 6.0, room temperature, after 4h standing; the volume ratio of two phases V PEG :V Dex 1:3, 1:2, 1:1, 2:1, 3:1;

[0046] Figure 4 Fluorescence microscope image of stable whole aqueous phase emulsion prepared by chitosan colloids at pH 6.0, room temperature, after 4h standing; the volume ratio of two phases V PEG :V Dex 1:3-3:1;

[0047] Figure 5 Fluorescence microscope image of stable whole aqueous phase emulsion prepared by chitosan colloids at pH 6.0, 55℃, after 4h standing; the volume ratio of two phases V PEG :V Dex 1:3, 1:2, 1:1, 2:1, 3:1;

[0048] Figure 6 Fluorescence microscope image of stable whole aqueous phase emulsion prepared by temperature-sensitive chitosan colloids at pH 6.0, 37℃, after 4h standing; the volume ratio of two phases V PEG :V Dex 1:3, 1:2, 1:1, 2:1, 3:1;

[0049] Figure 7 Fluorescence microscope image of stable whole aqueous phase emulsion prepared by temperature-sensitive chitosan colloids at pH 6.0, 45℃, after 4h standing; the volume ratio of two phases V PEG :V Dex 1:3, 1:2, 1:1, 2:1, 3:1;

[0050] Figure 8 Fluorescence microscope image of stable whole aqueous phase emulsion prepared by temperature-sensitive chitosan colloids at pH 6.0, 55℃, after 4h standing; the volume ratio of two phases V PEG :V Dex 1:3, 1:2, 1:1, 2:1, 3:1. DETAILED DESCRIPTION

[0051] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall into the scope of the present application.

[0052] The test materials and reagents used in the following examples, and the like, can be obtained from commercial channels unless otherwise specified.

[0053] If a specific technique or condition is not specified in the examples, the technique or condition described in the literature in the art or according to the product manual can be used.

[0054] Example 1

[0055] A preparation method of a temperature-sensitive chitosan colloid, comprising the following steps:

[0056] (1) Refining of N-isopropyl acrylamide (NIPAAM):

[0057] 40 g of NIPAAM was added to 100 mL of n-hexane at 60°C for dissolution. After complete dissolution, hot suction filtration was performed, and the filtrate was immediately poured into a beaker and placed in a fume hood for n-hexane volatilization.

[0058] (2) Recrystallization of azobisisobutyronitrile (AIBN) (note to avoid light):

[0059] 5 g of AIBN was poured into a beaker containing anhydrous ethanol at 40-50°C, stirred and dissolved, and then hot suction filtration was performed. The filtrate was immediately poured into another beaker and placed in a fume hood. After crystallization, the solid was obtained by filtration and dried in a vacuum drying oven at 40°C.

[0060] (3) Synthesis of carboxyl-terminated poly-N-isopropyl acrylamide (PNIPAAM-COOH):

[0061] 10 g of NIPAAM, 250 μL of mercaptopropionic acid (MPA), and 250 mg of AIBN were dissolved in 100 mL of isopropyl alcohol (IPA). Before polymerization, dry nitrogen was bubbled into the solution for 15 minutes. Then, the solution was placed in an oil bath at 75°C for 12 hours of reaction under an inert atmosphere (N2). After the reaction, the product was dialyzed in a cellulose membrane tube (MD: 3400) for three days with ultra-pure water, and the water was changed three times a day to remove impurities and unreacted substances. Finally, the product was freeze-dried to obtain PNIPAAM-COOH.

[0062] (4) Synthesis of chitosan-grafted poly-N-isopropylacrylamide:

[0063] At room temperature, using EDC / NHS as a condensing agent, terminal carboxyl-terminated poly(N-isopropylacrylamide) was grafted onto chitosan. The specific process was as follows: First, a deacetylated chitosan solution (0.1386 g) was prepared by uniform stirring in 100 mL of 1% acetic acid solution. Then, PNIPAAM-COOH (0.4283 g) was added to this solution. Subsequently, EDC (0.1398 g) / NHS (0.2864 g) (weight ratio 1:2) was added to the reaction mixture. The reaction was continuously stirred with a magnetic stirrer for 24 hours at room temperature. After dialyzing with distilled water using a cellulose membrane tube (MD: 3400) for three days with water changes three times daily, the product was freeze-dried and then lyophilized at -70°C and 2 Pa to obtain chitosan-grafted poly(N-isopropylacrylamide) (CS-g). - PNIPAAM).

[0064] (5) Thermosensitive chitosan colloidal particles (CS-g) - Preparation of PNIPAAM CPs solution:

[0065] Thermosensitive chitosan was dissolved in a 1% (v / v) acetic acid solution, and the pH of the solution was adjusted to 6.0 using NaOH solution, yielding a thermosensitive chitosan colloid concentration of 2.0 mg / mL. -1 .

[0066] Figure 1 The CS-g was prepared according to the method described in Example 1. - Transmission electron microscope image of PNIPAAM CPs; CS-g prepared - A single drop of PNIPAAM CPs solution was applied to a copper grid using a microsyringe and allowed to air dry at room temperature before being characterized by TEM. The graph shows that CS-g... - PNIPAAM CPs are spherical particles with a diameter of approximately 100-500 nm. Their particle size is sufficient to overcome the large interfacial thickness characteristic of all-aqueous emulsions.

[0067] One of the important factors affecting the stability of emulsions is the wetting ability of solid particle stabilizers, and the three-phase contact angle is one of the main means of characterizing the wetting performance of solid particles. Figure 2 It is CS-g - Contact angle images of PNIPAAM with PEG aqueous phase and Dex aqueous phase. The specific experimental procedure was as follows: PEG and Dex were dissolved in ultrapure water to prepare 20% (w / w) solutions. The PEG and Dex solutions were heated to 25°C, 37°C, 45°C, and 55°C, respectively. The CS-g prepared in Example 1 was then... -PNIPAAM was ground into powder, CS-g - PNIPAAM was adhered to the glass slide and flattened, and the CS-g prepared in Example 1 was characterized by a contact angle instrument - The three-phase contact angles of PNIPAAM with the heated PEG, DEX solution. From the contact angle data, it can be seen that the CS-g - The contact angles of PNIPAAM with the PEG phase and the Dex phase change with temperature, indicating that temperature can change its hydrophilicity and hydrophobicity, and also proving that the prepared temperature-sensitive chitosan glue can control the type of emulsion.

[0068] The above-mentioned application of the temperature-sensitive chitosan glue of Example 1 in regulating the type of water-in-water emulsion, the temperature-sensitive chitosan glue as an emulsifier for regulating the type of emulsion, and chitosan was compared, the present application used FITC-dextran to mark DEX, and observed under a fluorescence microscope, to explore the emulsion type at different temperatures, and the specific experiment was as follows:

[0069] At room temperature, 50 mg of CS-g - PNIPAAM was dissolved in 25 mL of 1% acetic acid solution, after complete dissolution, the pH value was adjusted to 6.0 using a NaOH solution on a pH machine to obtain 2 mg·mL -1 CS-g - PNIPAAM CPs solution. 6.25 g of polyethylene glycol (PEG, molecular weight 20000) was weighed and added to 2 mg·mL -1 CS-g - The beaker containing the PNIPAAM CPs solution was placed on a constant-temperature magnetic stirrer for stirring to fully dissolve, to obtain a CS-g - The mass fraction of the PNIPAAM CPs solution was 20wt% PEG solution; 6.25 g of dextran (Dex, molecular weight 200000) was weighed and added to the beaker containing the ultrapure water, a magnetic stirrer was placed in the beaker, and the beaker was placed on a constant-temperature magnetic stirrer for stirring to fully dissolve, and then the pH was adjusted to 6.0, and 1 mg of FITC was added for fluorescence labeling, to obtain a Dex solution with a mass fraction of 20wt%. The two aqueous solutions were mixed in a volume ratio V PEG :V Dex =1:3-3:1, and after mixing, the total volume was 6 mL. The PEG solution and the Dex solution were mixed in a 10 mL screw cap bottle, and a homogeneous speed of 8000 r·min -1 was used, and after 3 min of homogenization, a stable all-water phase emulsion was obtained. After the emulsion was placed in the dark for 4 h, it was observed under a fluorescence microscope as shown in Figure 3 V PEG :VDex = 1:3 is bicontinuous phase, V PEG :V Dex = 1:2, the emulsion type has phase transition (Dex becomes internal phase, PEG becomes external phase, i.e. Dex has larger volume but is internal phase).

[0070] Using chitosan as a control: 20wt% PEG solution: 50mg chitosan (CS, viscosity <200mp.s) was dissolved in 25mL, 1% acetic acid solution, and then the pH value was adjusted to 6.0 using NaOH solution to obtain 2mg.mL -1 CS CPs solution. 6.25g polyethylene glycol (PEG, molecular weight 20000) was added to the beaker containing 2mg.mL -1 CS CPs solution, and it was stirred on a constant temperature magnetic stirrer to fully dissolve, obtaining a PEG solution with a mass fraction of 20wt% dissolved in the CS CPs solution.

[0071] Control experiment 1: The above-prepared 20wt% Dex solution and 20wt% PEG solution dissolved in the CS CPs solution were mixed at room temperature, and the volume ratio of the two-phase aqueous solution was V PEG :V Dex = 1:3-3:1, and the total volume after mixing of the two phases was 6mL. The PEG solution and Dex solution were mixed in a 10mL screw bottle. At room temperature, the homogenization speed was 8000r.min -1 , and after homogenization for 3min, a stable all-aqueous phase emulsion was obtained. The emulsion was then placed in a water bath at room temperature and in the dark for 4h, and the emulsion type was observed under a fluorescence microscope, as shown in Figure 4 PEG :V Dex = 2:1, phase transition occurred (because the volume of PEG increased, it became the external phase).

[0072] Control experiment 2: The above-prepared 20wt% Dex solution and 20wt% PEG solution dissolved in the CS CPs solution were mixed, and the volume ratio of the two-phase aqueous solution was V PEG :V Dex = 1:3-3:1, and the total volume after mixing of the two phases was 6mL. The PEG solution and Dex solution were mixed in a 10mL screw bottle and heated to 55°C. At a temperature of 55°C, the homogenization speed was 8000r.min -1 , and after homogenization for 3min, a stable all-aqueous phase emulsion was obtained. The emulsion was then placed in a water bath at 55°C and in the dark for 4h, and the emulsion type was observed under a fluorescence microscope, as shown in Figure 5 PEG :V Dex = 2:1, phase transition occurred (because the volume of PEG increased, it became the external phase).​​

[0073] The chitosan colloids in control experiments 1 and 2 as emulsifiers of the whole aqueous phase emulsion, Figure 4 、 5 The temperature was changed, but the emulsion type did not change, and it was found that the unmodified chitosan could not change the emulsion type by changing the temperature. By comparison Figure 3 and Figure 4 It can be seen that the CS-g - PNIPAAM CPs as stabilizers, the V PEG :V Dex =1:2 (when the volume of Dex is large, as the internal phase) emulsion type is changed.

[0074] Example 2

[0075] Application of temperature-sensitive chitosan colloids in regulating the type of water-in-water emulsion, temperature-sensitive chitosan colloids as emulsifiers for regulating the type of emulsion, the specific process is:

[0076] The chitosan polymer colloids (CS-g - PNIPAAM CPs) prepared by the same method as in Example 1 were used to prepare whole aqueous phase emulsion, the specific preparation method is:

[0077] According to the same method as in Example 1, 20wt% PEG solution and 20wt% Dex solution were prepared. Two-phase aqueous solution was mixed in a volume ratio of V PEG :V Dex =1:3-3:1, the total volume was 6mL after mixing, the PEG solution and the Dex solution were mixed in a 10mL screw bottle, heated to 37℃. At 37℃, the homogenization speed was 8000r·min -1 , and a stable whole aqueous phase emulsion was obtained after homogenization for 3min. Then the emulsion was placed at 37℃ in the dark for 4h, and the emulsion type was observed under a fluorescence microscope. The emulsion type changed at V PEG :V Dex =1:1 (when the volume is the same, Dex becomes the internal phase), as shown in Figure 6 .

[0078] Example 3

[0079] Application of temperature-sensitive chitosan colloids in regulating the type of water-in-water emulsion, temperature-sensitive chitosan colloids as emulsifiers for regulating the type of emulsion, the specific process is:

[0080] The chitosan polymer colloids (CS-g - PNIPAAM CPs) prepared by the same method as in Example 1 were used to prepare whole aqueous phase emulsion, the specific preparation method is:

[0081] A 20 wt% PEG solution and a 20 wt% Dex solution were prepared using the same method as in Example 1. The two aqueous solutions were mixed at a volume ratio of V... PEG :V Dex The ratio of PEG solution to Dex solution is 1:3-3:1, and the total volume after mixing is 6 mL. The PEG solution and Dex solution are mixed in a 10 mL screw-top bottle and heated to 45°C in a water bath at a homogenization rate of 8000 r / min. -1 After homogenization for 3 minutes, a stable all-aqueous emulsion was obtained. The emulsion was then allowed to stand at 45°C in the dark for 4 hours, and the emulsion type was observed under a fluorescence microscope. The emulsion type was V... PEG :V Dex When the ratio is 1:1, it is a double continuous phase, V PEG :V Dex A phase transition occurs when the ratio is 2:1, such as... Figure 7 As shown.

[0082] Example 4

[0083] The application of thermosensitive chitosan colloids in regulating the type of water-in-water emulsions: The thermosensitive chitosan colloid acts as an emulsifier to regulate the type of emulsion. The specific process is as follows:

[0084] Chitosan polymer colloid (CS-g) was prepared using the same method as in Example 1. - PNIPAAM CPs) were used to prepare all-aqueous emulsions. The specific preparation method is as follows:

[0085] A 20 wt% PEG solution and a 20 wt% Dex solution were prepared using the same method as in Example 1. The two aqueous solutions were mixed at a volume ratio of V... PEG :V Dex The ratio of PEG solution to Dex solution is 1:3-3:1, and the total volume after mixing the two phases is 6 mL. The mixture is placed in a 10 mL screw-top bottle and heated to 55 °C in a water bath at a homogenization rate of 8000 r·min. -1 After homogenization for 3 minutes, a stable all-aqueous emulsion was obtained. The emulsion was then allowed to stand at 55°C in the dark for 4 hours, and the emulsion type was observed under a fluorescence microscope. The emulsion type was V... PEG :V Dex A phase transition occurs when the ratio is 2:1, such as... Figure 8 As shown.

[0086] contrast Figure 3 , Figures 6-8 It can be seen that the thermosensitive chitosan colloid of the present invention is thermosensitive, and can not only achieve phase inversion, but also achieve different volumes of phase inversion at different temperatures; under the same volume, different types of emulsions can be obtained by controlling different temperatures.

[0087] The above description of the embodiments is made for the purpose of enabling a person of ordinary skill in the art to understand and use the application. It is obvious that those skilled in the art can easily make various modifications to the embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present application is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. A method for preparing a temperature-sensitive chitosan colloid, characterized by, The preparation method comprises the following steps: 1) mixing N-isopropyl acrylamide, azobisisobutyronitrile and isopropyl alcohol, adding a chain transfer agent, stirring, heating the reaction under the atmosphere of nitrogen, dialyzing after the reaction is completed, and freeze-drying to obtain carboxyl-terminated poly-N-isopropyl acrylamide PNIPAM-COOH; 2) Dissolve chitosan in acetic acid solution to obtain chitosan solution, then add PNIPAM-COOH and condensing agent, stir and react at room temperature, after reaction, dialysis, freeze-drying, to obtain temperature-sensitive chitosan CS-g- PNIPAM - PNIPAM; 3) CS-g - The PNIPAM was dissolved in acetic acid solution, and the pH was adjusted to the appearance of Tyndall phenomenon by stirring, to obtain temperature-sensitive chitosan colloidal particles CS-g - PNIPAM CPs.

2. The production method according to claim 1, characterized by, In step 1), the mass ratio of the N-isopropyl acrylamide and azobisisobutyronitrile is 100:1-20:1; the ratio of the amount of azobisisobutyronitrile to isopropyl alcohol is 1:800-1:300 g / mL; the ratio of the amount of azobisisobutyronitrile added to the chain transfer agent is 3:1-1:3 mg / μL; and the chain transfer agent is mercaptopropionic acid.

3. The preparation method according to claim 1, characterized in that, In step 1), the heating reaction is at 50-80 ℃ for 8-15 h.

4. The method of claim 1, wherein, In step 2), the mass ratio of the chitosan and PNIPAM-COOH is 1:4-4:1; the mass ratio of the chitosan and the condensing agent is 1:4-4:1; the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC / NHS), and the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:3-3:

1.

5. A temperature-sensitive chitosan colloid prepared by the preparation method in any one of claims 1-4, wherein the particle size of the obtained colloid is 100-500 nm.

6. Use of the temperature-sensitive chitosan colloid according to claim 5 for the regulation of water-in-water emulsion types, characterized by, The temperature-sensitive chitosan colloid is used as an emulsifier for regulating the type of emulsion.

7. Use according to claim 6, characterized in that, Polyethylene glycol is dissolved in the temperature-sensitive chitosan colloid solution, and heated to a response temperature to obtain a polyethylene glycol solution; dextran is dissolved in ultrapure water, the pH value thereof is adjusted, and the dextran solution is heated to the same temperature as the polyethylene glycol solution; the polyethylene glycol solution and the dextran solution at the same temperature are mixed, and the mixture is left to stand until the mixture forms two macroscopic phases of layers; and the mixture is subjected to homogenization to form a whole-aqueous-phase emulsion.

8. Use according to claim 7, characterized in that, The concentration of polyethylene glycol in the polyethylene glycol solution is 10-30 wt%; the concentration of dextran in the dextran solution is 10-30 wt%; and the volume ratio of the polyethylene glycol solution to the dextran solution in the mixing is 1:3-3:

1.

9. Use according to claim 7 or 8, characterized in that, The temperature-sensitive chitosan colloidal solution is a temperature-sensitive chitosan colloidal solution obtained by dissolving temperature-sensitive chitosan colloids in an acetic acid solution with a volume fraction of 0.5-3.0%, and the concentration of the temperature-sensitive chitosan colloids is 0.5-5.0 mg·mL -1 .

10. Use according to claim 7 or 8, characterized in that, The adjustment of the pH value of the dextran solution and the heating to the same temperature as the polyethylene glycol solution are as follows: the temperature is 25-55 ℃, and the pH value is adjusted to 6.0 by using a NaOH solution.

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